Pathogenicity and Transmissibility of North American Triple Reassortant Swine Influenza A Viruses in Ferrets

Pathogenicity and Transmissibility of North AmericanTriple Reassortant Swine Influenza A Viruses in FerretsSubrata Barman1, Petr S. Krylov1, Thomas P. Fabrizio1, John Franks1, Jasmine C. Turner1, Patrick Seiler1,

David Wang1, Jerold E. Rehg2, Gene A. Erickson3, Marie Gramer4, Robert G. Webster1, Richard J. Webby1*

1 Division of Virology, Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America, 2 Department of

Pathology, St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America, 3 Veterinary Diagnostic Laboratory (NCVDL) System, North Carolina

Department of Agriculture, Raleigh, North Carolina, United States of America, 4 Veterinary Diagnostic Laboratory, College of Veterinary Medicine, University of Minnesota,

St. Paul, Minnesota, United States of America

Abstract

North American triple reassortant swine (TRS) influenza A viruses have caused sporadic human infections since 2005, buthuman-to-human transmission has not been documented. These viruses have six gene segments (PB2, PB1, PA, HA, NP, andNS) closely related to those of the 2009 H1N1 pandemic viruses. Therefore, understanding of these viruses’ pathogenicityand transmissibility may help to identify determinants of virulence of the 2009 H1N1 pandemic viruses and to elucidatepotential human health threats posed by the TRS viruses. Here we evaluated in a ferret model the pathogenicity andtransmissibility of three groups of North American TRS viruses containing swine-like and/or human-like HA and NA genesegments. The study was designed only to detect informative and significant patterns in the transmissibility andpathogenicity of these three groups of viruses. We observed that irrespective of their HA and NA lineages, the TRS viruseswere moderately pathogenic in ferrets and grew efficiently in both the upper and lower respiratory tracts. All NorthAmerican TRS viruses studied were transmitted between ferrets via direct contact. However, their transmissibility byrespiratory droplets was related to their HA and NA lineages: TRS viruses with human-like HA and NA were transmitted mostefficiently, those with swine-like HA and NA were transmitted minimally or not transmitted, and those with swine-like HAand human-like NA (N2) showed intermediate transmissibility. We conclude that the lineages of HA and NA may play acrucial role in the respiratory droplet transmissibility of these viruses. These findings have important implications forpandemic planning and warrant confirmation.

Citation: Barman S, Krylov PS, Fabrizio TP, Franks J, Turner JC, et al. (2012) Pathogenicity and Transmissibility of North American Triple Reassortant SwineInfluenza A Viruses in Ferrets. PLoS Pathog 8(7): e1002791. doi:10.1371/journal.ppat.1002791

Editor: Andrew Pekosz, Johns Hopkins University - Bloomberg School of Public Health, United States of America

Received December 8, 2011; Accepted May 22, 2012; Published July 19, 2012

Copyright: � 2012 Barman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and HumanServices (Contracts No. HHSN266200700005C and HHSN266200700007C), and by the American Lebanese Syrian Associated Charities (ALSAC). The funders had norole in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: Dr. Webster receives funding from F. Hoffmann LaRoche Ltd., but the funds were not used in support of the research presented in themanuscript. This does not alter our adherence to all PLoS Pathogens policies on sharing data and materials.

* E-mail: [email protected]

Introduction

For nearly 70 years, swine influenza virus in North America was

relatively stable, dominated by the classical-swine H1N1 (cH1N1)

subtype [1]. However, H3 seasonal human influenza A viruses

were circulating at low frequency in U.S. swine [2]. In 1998,

influenza epidemiology in North American swine changed

dramatically with the emergence of double-reassortants (combin-

ing gene segments of cH1N1 and seasonal human H3N2 influenza

A viruses) and triple-reassortants (adding gene segments from

avian influenza lineages). The triple-reassortants gained predom-

inance in North American swine and continued to evolve, further

reassorting with cH1N1 and contemporary seasonal human

influenza viruses [3,4]. All of the currently circulating North

American triple-reassortant swine (TRS) influenza A viruses

contain a similar constellation of internal genes (avian PA and

PB2, human PB1, and classical swine-lineage M, NP, and NS), but

their surface glycoproteins are derived from different lineages

(classical swine-lineage H1 and N1 and seasonal human-lineage

H1, H3, N1 and N2).

Sporadic infections with TRS H1N1 (swine-like HA and NA)

and H1N2 (swine-like HA, human-like NA) viruses have been

reported in humans exposed to swine in North America [5]. Some

have included severe lower respiratory tract disease and diarrhea.

H3N2 (human-like HA and NA) TRS viruses have also been

isolated from humans [6,7,8]. In 2009, TRS viruses with human-

like H1 and N1 (closely related to A/Brisbane/59/2007 [H1N1])

caused cough, fever, nasal congestion, rhinorrhea, sneezing,

malaise, and dizziness in humans [9]. These symptoms were very

similar to those caused by the 2009 H1N1 pandemic viruses,

which possessed six gene segments (PB2, PB1, PA, HA, NP, and

NS) closely related to those of North American TRS viruses [10].

However, unlike the 2009 H1N1 pandemic viruses, the TRS

viruses were not reported to be transmissible among humans.

Despite extensive recent studies of the pathogenicity and

transmissibility of pH1N1 viruses in different animal models

[11–14], there is very little information of this kind about North

American TRS viruses. A/swine/Kansas/77778/2007 (H1N1), a

triple reassortant similar to H1N1 viruses that infected humans

and pigs at an Ohio county fair in 2007, was isolated from swine

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herds in the Midwestern United States. This virus is highly virulent

in swine and is readily transmitted to sentinel pigs [15]. TRS virus

A/Swine/Texas/4199-2/98 (H3N2) was also shown to be

transmissible from infected swine to direct-contact swine and

from them to a second group of direct-contact swine [16]. Belser

and co-workers found two North American H1N1 TRS viruses

(with swine-like HA and NA) isolated from humans to be

pathogenic in mice [17]. In ferrets, these viruses showed

pathogenicity similar to that of 2009 pandemic H1N1 influenza

virus but less efficient transmissibility [18]. We have shown that

the TRS virus A/swine/Arkansas/2976/02 (H1N2) and the

Eurasian avian-like swine virus A/swine/Hong Kong/NS29/09

(H1N1) are not transmissible via respiratory droplets in ferrets

[19]. The TRS virus A/swine/Guangdong/1222/2006 (H1N2)

and the Eurasian avian-like swine virus A/swine/Fujian/204/

2007 (H1N1) were recently shown not to be transmissible by direct

contact in guinea pigs [20]. Very recently, Pearce et al.

demonstrated that H3N2 TRS viruses isolated from humans were

efficiently transmitted via respiratory droplets (RD) in ferrets [8].

Most of the North American TRS viruses belong to three

subtypes: H1N1, H1N2, and H3N2; H1 and N1 are of the

classical swine or seasonal human lineages, while H3 and N2 are

of seasonal human lineage only. TRS viruses with human-like HA

and NA have recently become the predominant influenza viruses

isolated from swine in the US (St. Jude swine influenza surveillance

program, unpublished data), but their transmissibility has not been

tested in the ferret model. Ferrets are an established small-animal

model that appears to recapitulate the pathogenicity and

transmissibility of human seasonal influenza A viruses [11–13]

and the poor human transmissibility of H5 and H7 avian influenza

A viruses [21–23].

In the present study, we used the ferret model to evaluate the

pathogenicity and transmissibility of three distinct groups of North

American TRS viruses (H1N1 viruses with classical swine-like HA

and NA; H1N2 viruses with classical swine-like HA but human-

like NA; and H1N1, H1N2, and H3N2 viruses with human-like

HA and NA) and of the Eurasian avian-like swine virus A/sw/

Italy/1369-7/1994 (H1N1) (Italy/94). Because a limited number

of ferrets could be used, the study was designed to detect patterns

in the transmissibility and pathogenicity of TRS viruses that, once

confirmed, will have important implications for pandemic

preparedness. Italy/94 virus was less efficiently transmissible than

the North American TRS viruses. The North American TRS

viruses, regardless of their HA and NA lineages, were readily

transmissible to co-housed (direct contact, DC) ferrets, while

viruses with human-like HA and NA or with human-like NA alone

showed enhanced transmission via respiratory droplets.

Results

PathogenicityFerrets inoculated with 106 pfu of each virus (a dose previously

found to result in consistent infection [11–13,18,22]) showed only

mild clinical signs of illness, mild to moderate weight loss (,3% to

9%), and infrequent sneezing (Table 1). Among the ferrets that lost

weight, weight loss was maximal during days 4 to 6 pi, and then

weight began to increase. Ferrets that did not lose weight showed

no significant change until day 4 pi and then began gaining

weight. A few inoculated ferrets (e.g., two inoculated with Italy/94)

gained weight continuously, starting on day 1. A few ferrets

(mainly those inoculated with TRS viruses with human-like HA

and NA) had elevation of body temperature (maximum increase,

1.5uC). We observed no significant lethargy and no ruffled fur.

Infectious virus was observed in nasal washes until approximately

day 6 post-inoculation (p.i.), with peak titers on day 2 p.i. TRS

virus A/sw/NC/47834/2000 (H1N1, with swine-like HA and

NA) caused the least nasal virus shedding (mean peak titer, 56104

pfu/ml vs. 106 pfu/ml for the other two viruses in this group)

(Fig. 1).

One of the TRS isolates (A/sw/IN/9K035/1999 [H1N2]) with

swine-like HA but human-like NA caused peak nasal virus

shedding (mean peak titer, 106 pfu/ml) substantially higher than

that caused by the other two viruses in this group (mean peak

titers, 26105 pfu/ml and 16105 pfu/ml). TRS isolates containing

human-like HA and NA caused the highest peak nasal wash titers

(mean, 106 pfu/ml). Infectious virus particles were identified at

various titers in the lungs of all ferrets inoculated with the studied

TRS viruses (Table 1). Ferrets inoculated with the Italy/94 virus

had low peak nasal wash titers (mean, 1.76105 pfu/ml) and

exhibited almost no clinical signs. However, infectious virus

particles were obtained from the lungs of both inoculated ferrets.

These findings show that overall, North American TRS viruses

grow efficiently in both the upper and lower respiratory tracts of

ferrets irrespective of their HA and NA lineages and cause

moderate pathogenicity, similar to the reported pathogenicity of

the 2009 pandemic H1N1 viruses [11–14].

Lung histopathologyAll of the North American TRS viruses caused bronchitis,

bronchiolitis, alveolitis and alveolar wall interstitial changes, with

varying degrees of involvement and severity. The degree of

involvement and the severity also varied to some extent in different

ferrets inoculated with the same virus and in different lobes of the

same lung. Fig. 2 shows representative changes. The bronchitis

featured intraluminal granulocytes and/or mucus, bronchial

epithelial hyperplasia with submucosal mucus gland loss, and

mixed inflammatory-cell infiltrates. The bronchiolitis featured

intraluminal cellular debris, sloughed epithelial cells, and inflam-

matory cells (macrophages and/or granulocytes) with or without

bronchiolar epithelial cell necrosis and/or regenerative epithelial

cell hyperplasia and hypertrophy. In the alveolitis, the alveoli

surrounding the bronchioles contained a mixture of inflammatory

cell infiltrates (granulocytes, lymphocytes, plasma cells and

macrophages) and foci of pneumocyte hyperplasia. The interstitia

(alveolar walls) were either normal or thickened by increased

Author Summary

North American triple reassortant swine (TRS) influenza Aviruses have caused sporadic human infections, buthuman-to-human transmission has not been established.We wished to elucidate potential human health threatsposed by the TRS viruses and to identify determinants ofvirulence in the TRS and closely related 2009 H1N1pandemic viruses. We used a ferret model to evaluatethe pathogenicity and transmissibility of North AmericanTRS viruses with the HA and NA antigenic proteins ofswine viruses and of human viruses. We observed that theNorth American TRS viruses grew efficiently in both theupper and lower respiratory tracts and caused moderatepathogenicity in ferrets. The viruses were readily transmis-sible via direct contact, irrespective of their HA and NAlineages. However, transmissibility via respiratory dropletswas substantially greater when the viruses carried the HAand NA of human influenza A viruses rather than of swineinfluenza A viruses. Because ferrets are a useful model ofhuman influenza infection, this finding helps to predictfeatures that increase the risk to human health.

North American TRS Influenza A Viruses in Ferrets

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cellularity. Italy/94 virus caused similar morphologic changes but

they were far less severe than those caused by the North American

TRS viruses.

TransmissionUnlike the 2009 H1N1 pandemic viruses, North American TRS

viruses are not known to be transmissible among humans [5,9]. To

investigate factors that affect transmissibility, we assessed the

transmission of different TRS viruses by direct contact (DC; in co-

housed ferrets) and by respiratory droplets (RD). Italy/94

(Eurasian avian-like swine) virus was transmitted to only one of

two DC ferrets and to neither RD ferret (Table 2, Fig. 1B),

indicating poor transmission efficiency.

Among the TRS viruses with swine-like HA and NA, A/sw/NC/

47438/2000 (H1N1), which had the lowest mean peak nasal wash

titer (#105 pfu/ml), was transmitted only by direct contact (2/2 DC,

0/2 RD) (Fig. 1C). Although the A/sw/NC/18161/2002 virus

replicated efficiently (mean peak nasal wash titer, 106 pfu/ml) in

donor ferrets, transmission was observed in only one of two DC

ferrets and in neither RD ferret. Infectious A/sw/MN/6998/2003

was detected on day 7 post-exposure (p.e.) (day 8 p.i.) in the nasal

wash of one of the two RD ferrets. Overall, only one of the three

viruses in this group was transmitted via RD, to one of two ferrets;

the remaining two viruses were not transmitted via RD. Therefore,

TRS viruses with swine-like HA and NA were poorly transmitted

via RD. Poor RD transmission of TRS viruses containing swine-like

HA and NA has been reported previously [16].

The TRS viruses with human-like HA and NA replicated

efficiently (mean peak nasal wash titer, ,106 pfu/ml) and were

efficiently transmitted (2/2 DC, 2/2 RD) in ferrets (Fig. 1E and

Table 2), consistent with a recent report on the RD transmissibility

of TRS viruses containing human-like HA and NA (H3N2) [8].

The transmission efficiency of TRS viruses with swine-like HA and

human-like NA (N2) varied from strain to strain. Among these

viruses, A/sw/MN/1182/2001 was as efficiently transmitted (2/2

DC, 2/2 RD) as TRS viruses with human-like HA and NA. In

contrast, infectious A/sw/MN/5763/2003 and A/sw/IN/

9K035/1999 viruses were detected in the nasal wash of only

one of two RD ferrets (Fig. 1D, Table 2). A/sw/MN/5763/2003

virus was first detected in a RD ferret on day 7 p.e., while the other

two viruses in this group were first detected on day 3 p.e.

Taken together, these results suggest that in the ferret model, 1)

transmissibility of Italy/94 (Eurasian avian-like swine) virus is

poor, 2) North American TRS viruses are readily transmissible by

direct contact irrespective of their HA and NA lineages, and 3)

transmissibility via RD of TRS viruses with swine-like HA and

NA, swine-like HA but human-like NA, and human-like HA and

NA is poor, moderate, and efficient, respectively.

Growth kinetics in MDCK cellsOur results show that unlike seasonal H1N1 viruses, whose

replication is reported to occur primarily in the upper respiratory

tract [11–13], TRS viruses grow efficiently in ferret lungs and

cause substantial lung pathology, similar to that reported for

pandemic H1N1 viruses [11–13]. As the temperature is higher in

the lower than the upper respiratory tract, ability to grow at a

higher temperature might favor virus growth in the ferret lung. To

better understand the pathogenicity of the swine isolates studied,

we examined the multi-cycle growth kinetics of these swine viruses,

of seasonal human H1N1 virus A/Brisbane/59/2007, and of the

2009 pandemic H1N1 virus A/Mexico/4482/2009 in MDCK

cells at different temperatures. MDCK cell monolayers were

inoculated with the viruses at a multiplicity of infection (MOI) of

0.001 and incubated at 33uC, 37uC, and 39.5uC. At different h

p.i., supernatants were harvested and virus was titrated by pfu

assay (Fig. 3). At 33uC, Italy/94 (Eurasian avian-like swine) virus

and TRS viruses with swine-like HA and NA had similar growth

kinetics, with peak titers of ,108 pfu/ml. TRS viruses with

Table 1. Clinical signs and virus replication in inoculated ferrets.

Viruses Clinical signs Virus titer (log10 pfu/ml)b

Weight loss No/total(meana max. % loss)

Sneezing(No/total) Nasal turbinates Trachea upper Trachea lower Lungc

Eurasian avian like swine:

A/sw/Italy/3169-7/1994 (H1N1) 1/4 (5) 0/4 –, – –, 5.3 –, 4.5 3.3, 4.6

TRS with sw-like HA and NA

A/sw/NC/47834/2000 (H1N1) 2/4 (5) 0/4 4.6, 4.7 3.7, 2.8 3.2, 2.3 3.5, 2.9

A/sw/NC/18161/2002 (H1N1) 2/4 (9) 1/4 4.6, – 3.5, – 2.8, 1.7 3.5, 2.6

A/sw/MN/6998/03 (H1N1) 2/4 (6) 1/4 2.9, 3.9 4.8, 4.5 4.9, 5.0 6.0, 2.6

TRS with sw-like HA and hu-like NA

A/sw/IN/9K035/99 (H1N2) 3/4 (5) 2/4 4.7, 5.1 2.6, 5.3 3.8, 4.2 5.3, 5.3

A/sw/MN/1192/01 (H1N2) 2/4 (9) 2/4 4.5, 1.3 5.6, 4.1 4.7, 5.5 1.8, 6.0

A/sw/MN/5763/03 (H1N2) 1/4 (3) 1/4 5.0, 4.7 4.5, 2.6 3.8, 2.8 3.5, 2.3

TRS with hu-like HA and NA

A/sw/NC/38448-1/2005 (H1N1) 1/4 (5) 1/4 5.7, 2.9 4.8, 3.2 4.3, 3.8 5.2, 4.7

A/sw/OK/011521-5/2008 (H1N2) 2/4 (5) 2/4 5.3, – 5.7, 6.3 6.0, 5.8 5.1, 5.8

A/sw/OK/011506/2007 (H3N2) 1/4 (7) 0/4 5.1, 4.8 4.2, 2.9 4.5, 2.8 5.1, 3.7

Sw, swine; hu, human.–, Below the limit of detection (1.3 log10 pfu/ml).aMean (when multiple ferrets lost weight) percent maximum weight loss (ferrets not showing weight loss were excluded from this calculation).bValues derived from two ferrets.cHomogenates combining portions of all six lobes.doi:10.1371/journal.ppat.1002791.t001

North American TRS Influenza A Viruses in Ferrets

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Figure 1. Transmissibility of North American TRS viruses in ferrets. A) Isolator scheme. Four donor ferrets were inoculated with 106 pfu ofvirus and housed in the lower cages. The next day, two donor ferrets were moved into separate cages, each containing one naı̈ve DC ferret. Twonaı̈ve RD ferrets were housed separately in cages adjacent to the donor ferrets but separated by grills to allow unobstructed airflow while preventingdirect contact. Nasal washes were collected from inoculated (red and gray bars) and contact (green, DC; blue, RD) animals on the indicated days p.i.

North American TRS Influenza A Viruses in Ferrets

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human-like HA and NA grew to substantially higher titers (,109

pfu/ml), while TRS viruses with swine-like HA and human-like

NA grew to titers similar to those of seasonal H1N1 or 2009

pH1N1 viruses (108–109 pfu/ml, Fig. 3). At 37uC, although final

yield increased only slightly, replication of all viruses was

accelerated, as noted by significantly higher titers at 12 and 18 h

p.i. At 39.5uC, replication of all swine viruses and of the 2009

pH1N1 virus A/Mexico/4482/2009 was less (by a factor of 10 to

100) than their replication at 37uC. However, the reduction of

virus titer at 39.5uC was greatest for seasonal human H1N1 virus

A/Brisbane/59/2007 (66103 pfu/ml vs. 106–108 pfu/ml for swine

and pH1N1 viruses).

At all three temperatures, TRS viruses with human-like HA and

NA grew to the highest titers, whereas those with swine-like HA

and NA grew to the lowest titers. These replication characteristics

somewhat paralleled the viruses’ overall respiratory droplet

transmission efficiency.

Discussion

Despite sporadic human infections with North American TRS

influenza A viruses, their human-to-human transmission has not

been established, and pathogenicity and transmission studies in

animal models have been very limited. This study of the

for virus titration. Inoculated animals remaining in the lower cages were euthanized on day 5 p.i. for tissue studies (nasal washes were collected ondays 2 and 4 p.i.). B–E) Nasal wash titers of B) Eurasian avian-like swine, C) TRS viruses with sw-like HA and NA, D) TRS viruses with sw-like HA but hu-like NA, and E) TRS viruses with hu-like HA and NA. Color coding is shown in panel A. Day 1 p.i. = day 0 post-exposure. sw, swine; hu, human.doi:10.1371/journal.ppat.1002791.g001

Figure 2. Histopathology of ferret lung tissue. Lung tissue of control (un-inoculated) and virus-inoculated ferrets was collected on day 5 p.i.Formalin-fixed, paraffin-embedded 5-mm sections were stained with hematoxylin and eosin and microscopically examined in a blinded fashion.Representative images show bronchi (A–D), bronchioles (E–H), and alveoli (I–L) from un-inoculated (A,E,I) and virus-inoculated ferrets. The two NorthAmerican TRS viruses (C,G,K and D,H,L) caused bronchitis, bronchiolitis, alveolitis, and alveolar wall interstitial changes. The bronchitis featuredintraluminal granulocytes and/or mucus, bronchial epithelial hyperplasia with submucosal mucus gland loss, and mixed inflammatory-cell infiltrates.The bronchiolitis featured intraluminal cellular debris, sloughed epithelial cells, and inflammatory cells (macrophages and/or granulocytes). Theperibronchiolar alveoli contained mixed inflammatory cell infiltrates and foci of pneumocyte hyperplasia. The Eurasian avian-like swine virus (B,F,J)caused morphologic changes similar to those caused by the TRS viruses but far less severe.doi:10.1371/journal.ppat.1002791.g002

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pathogenicity and transmissibility of North American TRS viruses

containing both swine- and human-like HA and NA found that the

viruses grow efficiently in both the upper and lower respiratory

tracts and cause moderate pathogenicity similar to that reported

for the 2009 pandemic H1N1 viruses [11–14]. The TRS viruses

were readily transmissible by direct contact in ferrets, irrespective

of their HA and NA lineages. However, RD transmissibility varied

significantly with the lineages of HA and NA: TRS viruses with

swine-like HA and NA, swine-like HA but human-like NA, and

human-like HA and NA were transmitted poorly, moderately, and

efficiently, respectively, via respiratory droplets.

Multiple viral factors, including HA receptor specificity, human-

specific amino acid residues (e.g., 627K/701N) in PB2, and

balance between HA and NA, are known to influence transmission

and pathogenicity in humans [24–28]. Like the 2009 pH1N1

viruses, all TRS viruses studied here have avian-origin PB2

containing 627E and 701D. However, SR polymorphism (590S

and 591R) within pH1N1 PB2 was shown to partly compensate

for the absence of 627K in polymerase activity and virus

replication in human A549 cells, suggesting that this polymor-

phism plays a role in efficient growth of pH1N1 viruses in the

human upper respiratory tract [29]. E627K substitution in PB2

was later shown not to alter the growth of pH1N1 virus in MDCK

cells at 33uC, 37uC, or 39uC or to significantly alter its virulence

and replication in mouse and ferret lung tissues [30–32].

Importantly, the PB2 of Italy/94 (Eurasian avian-like swine) virus,

containing 627E/701D, was associated with less lung pathology

and transmissibility; it also lacks the SR polymorphism, instead

containing 590G/591Q, which were shown to reduce the

polymerase activity of 2009 pH1N1 virus by 50% [29]. Like

pH1N1, all of our TRS viruses contain the avian-specific amino

acids 627E and 701D and the SR polymorphism (590S and 591R)

in PB2, (with the exception of A/sw/NC/47834/2000, which

contains 590S and 591Q and showed lower nasal wash titers and

poor transmissibility in ferrets), indicating involvement of other

factors in their differential RD transmission. In our experiments,

although the TRS viruses with human-like HA and NA replicated

efficiently (mean peak nasal wash titer, ,106 pfu/ml) and were

efficiently transmitted in ferrets (Fig. 1E and Table 2), transmission

did not always parallel virus shedding. For example, the TRS virus

A/sw/NC/18161/2002 (H1N1), with swine-like HA and NA, had

an average peak nasal wash virus titer (106 pfu/ml) similar to those

of viruses with human HA and/or NA but was least transmissible

(1/2 DC, 0/2 RD) in ferrets. In contrast, the TRS virus A/sw/

MN/1192/2001(H1N2), with swine-like HA and human-like NA,

had a significantly lower average peak nasal wash virus titer

(26105 pfu/ml) than A/sw/NC/18161/2002 (106 pfu/ml) but

was transmitted efficiently in ferrets.

H1 HA of North American swine isolates comprises four distinct

phylogenetic groups, H1a (cH1N1), H1b (TRS H1N1-like), H1c(TRS H1N2-like), and H1d (human-like) [33,34]. The HAs of

some recent TRS H1N1 isolates with swine-like HA and NA are

closely related to H1c (Fig. S1), as are pH1N1 HAs. Recently, two

TRS viruses isolated from humans, A/Texas/14/08 (H1N1, H1b)

and A/Ohio/2/07 (H1N1, H1c), showed poor aerosol transmis-

sibility in ferrets [18]. In our study, RD transmission of H1N1

TRS viruses with swine-like H1b HA and swine-like NA was poor

in ferrets. However, the H1N2 TRS viruses A/sw/MN/1182/

2001 (H1N2) and A/sw/IN/9K035/1999 (H1N2) which possess

H1c HA, were readily transmitted via respiratory droplets,

suggesting that human-like NA (N2) is responsible for RD

transmissibility of North American TRS viruses containing

swine-like H1c HA. However, studies using reassortant RG

viruses are needed to confirm that human-like NA (N2) can

enhance the RD transmissibility of TRS viruses containing swine-

like HA. Our group and, more recently, others have demonstrated

that pH1N1 NA and M can enhance RD transmission of TRS

viruses in ferrets [19,35]. However, others have shown that in a

guinea pig model that Eurasian avian-like swine virus (A/sw/

Fujian/204/2007[H1N1]) NA and M are not sufficient to alter the

Table 2. Transmission of North American TRS viruses in ferrets via direct contact (co-housing) and respiratory droplets.

Viruses Transmission (No. positive/total)

Direct contact Respiratory droplets

Virus detectiona Seroconversion Virus detectiona Seroconversion

Eurasian avian like swine:

A/sw/Italy/3169-7/1994 (H1N1) 1/2 1/2 0/2 0/2

TRS with sw-like HA and NA

A/sw/NC/47834/2000 (H1N1) 2/2 2/2 0/2 0/2

A/sw/NC/18161/2002 (H1N1) 1/2 1/2 0/2 0/2

A/sw/MN/6998/03 (H1N1) 2/2 2/2 1/2b 1/2

TRS with sw-like HA and hu-like NA

A/sw/IN/9K035/99 (H1N2) 2/2 2/2 1/2 1/2

A/sw/MN/1192/01 (H1N2) 2/2 2/2 2/2 2/2

A/sw/MN/5763/03 (H1N2) 2/2 2/2 1/2b 1/2

TRS with hu-like HA and NA

A/sw/NC/38448-1/2005 (H1N1) 2/2 2/2 2/2 2/2

A/sw/OK/011521-5/2008 (H1N2) 2/2 2/2 2/2 2/2

A/sw/OK/011506/2007 (H3N2) 2/2 2/2 2/2 2/2

Sw, swine; hu, human.aIn nasal wash specimens.bDelayed transmission: virus detected on day 7 post-exposure (day 8 p.i.).doi:10.1371/journal.ppat.1002791.t002

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non-transmissibility of North American TRS virus (A/sw/

Guandong/1222/2006[H1N2]) and that the HA and NS of

2009 pandemic H1N1 virus (A/Beijing/7/2009) contributes to its

transmissibility [20].

We found that the North American TRS viruses grew well at

39.5uC. The TRS viruses yielded 26106 to 26108 pfu/ml at

39.5uC, while seasonal human H1N1 A/Brisbane/59/07 yielded

only 66103 pfu/ml under identical growth conditions (Fig. 3). This

finding may explain the growth of TRS viruses in ferret lungs. At

33uC and 37uC, the yield of seasonal human virus (26108 pfu/ml)

and TRS viruses was similar. Interestingly, at all three temper-

atures the growth kinetics of the TRS viruses with swine-like HA

was very similar to that of the 2009 pandemic A/Mexico/4482/

2009 (H1N1) virus (Fig. 3). It was reported that unlike seasonal

H1N1 viruses, whose replication is primarily restricted to the

upper respiratory tract, the 2009 pandemic H1N1 viruses

replicated efficiently in ferret lungs [11–13]. A recent study found

that replacing the HA of seasonal H1N1 virus A/New York/312/

2001 with the HA of 2009 pH1N1 A/Mexico/4108/2009 (swine-

like) virus reduced surfactant protein D binding and increased lung

pathology in mice, although it did not increase lung virus titers

[36]. In our experiments, TRS viruses with either swine-like or

human-like HA caused significant lung pathology, yielded high

lung virus titers, and replicated efficiently in MDCK cells at

39.5uC. As the lower respiratory tract is warmer, ability to grow at

a higher temperature may be responsible at least in part for the

efficient lung growth and significant lung pathology in ferrets.

Further studies of growth characteristics in primary human

respiratory epithelial cells, which may more closely recapitulate

the human respiratory tract, are warranted. The molecular

Figure 3. Growth characteristics of North American TRS viruses in MDCK cells. Cells were inoculated with the respective viruses at an MOIof 0.001 and incubated at the indicated temperatures. At the indicated h p.i., supernatants were harvested and virus was titrated by pfu assay. At thehigher temperatures (37uC and 39.5uC), cytopathic effects caused detachment of most infected cells from the dish after 38 h p.i.; therefore, virusrelease was not examined beyond that point. Values are the mean of two independent experiments performed in duplicate (n = 4).doi:10.1371/journal.ppat.1002791.g003

North American TRS Influenza A Viruses in Ferrets

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determinants of the efficient in vitro growth of the TRS and pH1N1

viruses at 39.5uC and the relation of this growth to their efficient

replication in ferret lungs are of interest for future studies.

TRS viruses containing human-like HA and NA showed the

highest RD transmissibility in the ferret model, likely reflecting a

higher rate of replication (high virus titers in vitro and in vivo),

efficient release of progeny virions in the presence of human-like

NA, and efficient re-infection in the presence of human-like HA.

In contrast, this group of TRS viruses causes only sporadic human

infection, indicating a possible limitation of the ferret model.

Importantly, however, ferrets used in this study (and in all

transmissibility studies) were farm-raised and sero-negative for

influenza A viruses. Acquired immunity within the human

population, in addition to viral and environmental factors, plays

a critical role in human-to-human transmission of influenza A

viruses [37]. It is possible that vaccination and pre-exposure to

human-like HA and NA in the human population inhibits the

spread of this group of viruses in humans. Unlike HA-specific

antibodies, NA-specific antibodies do not prevent influenza virus

infection, and NA immunity is referred to as infection-permissive

[38]. However, humoral immunity induced by NA can markedly

reduce virus replication and release, moderating the severity and

duration of illness [39–42]. Human infections with H1N2 TRS

viruses containing swine-like HA have been reported [5], but in

humans, unlike the ferret experimental model, transmission is

likely to be partially inhibited by NA-mediated immunity to

seasonal influenza viruses, including H3N2. Therefore, the rapid

worldwide human spread of pH1N1 may be partially explained by

its acquisition of Eurasian avian-like swine virus NA and M in a

North American TRS genetic background (with swine-like HA) in

two ways. First, its RD transmissibility could have been enhanced

by the presence of the Eurasian NA and M and second, its

pandemic potential could have been enhanced by the absence of

immunity to the swine-like HA and NA in the human population.

The pandemic 2009 H1N1 virus is now the predominant

human H1N1 influenza virus worldwide. Vaccine against seasonal

human H1N1 does not offer significant protection against 2009

pH1N1, and therefore seasonal H1N1 has been replaced by 2009

pH1N1 (with North American swine-like HA and Eurasian swine-

like NA) in the World Health Organization’s recommended

trivalent vaccine. TRS viruses are reported to cause severe lower

respiratory tract disease and diarrhea in humans [5]. Here we

have shown that unlike seasonal H1N1 (e.g., A/Brisbane/59/

2007), whose replication is reported to be restricted primarily to

the upper respiratory tract [11], TRS viruses grow efficiently in

the lung and cause substantial lung pathology in ferrets. Most

importantly, we have shown that H1N1 TRS viruses with human-

like HA and NA (which reportedly do not cross-react with

antibody to 2009 pandemic H1N1 [43]) are efficiently transmitted

in the ferret model, indicating that in the absence of pre-exposure

or vaccination to seasonal H1N1, these viruses may be transmis-

sible among humans, especially young children, and therefore are

a public health concern.

Materials and Methods

AnimalsFour- to six-month-old male ferrets (Triple F farms, Sayre, PA;

Marshall Farms, Hazle Township, PA) that were serologically

negative for currently circulating influenza viruses by hemagglu-

tination inhibition (HI) assay were used. All animal experiments

were conducted in an Animal Biosafety Level 2+ (level 2 with

enhanced biocontainment for pandemic H1N1 influenza A virus)

facility at St. Jude Children’s Research Hospital, in compliance

with the policies of the National Institutes of Health and the

Animal Welfare Act and with the approval of the St. Jude

Children’s Research Hospital Animal Care and Use Committee.

Cells and virusesMDCK cells were maintained in Dulbecco modified Eagle’s

medium (DMEM; Invitrogen Corporation, Grand Island, NY)

supplemented with 10% fetal bovine serum and antibiotics-

antimycotic (Sigma, St. Louis, MO; 100 U/ml penicillin, 100 mg

streptomycin, and 0.25 mg amphomycin per ml). Stock viruses

were propagated in embryonated chicken eggs at 37uC for 48 h.

All isolates underwent a limited number of passages in eggs to

maintain their original properties. The genome sequences of A/

sw/OK/011521-5/2008 (H1N2), A/sw/OK/011506/2007

(H3N2), A/sw/IN/9K035/99 (H1N2), and A/Mexico/4482/

2009 (H1N1) have been described previously [43–45]. Our group

previously performed genome sequencing and GenBank submis-

sion of A/Brisbane/59/2007 (H1N1). For all other viruses,

complete genomes were sequenced as described previously [46]

and sequences were submitted to GenBank. Sequence alignment

and phylogenetic analysis of HA and NA (Fig. S1) and the other

six gene segments (data not shown) confirmed Italy/94 (H1N1) as

a Eurasian avian-like swine virus and the other nine swine viruses

as North American triple reassortants of the subtypes and HA and

NA lineages shown in Table 1.

In vitro virus growth kinetics and plaque assayMDCK cell monolayers were inoculated with viruses at an

MOI of 0.001 and maintained in virus growth medium (modified

Eagle’s medium [Invitrogen] containing 1% BME vitamins

[Sigma], 0.2% BSA [Calbiochem], 1.6 mg/ml NaHCO3 [Invitro-

gen], antibiotics-antimycotic [Sigma], and 1.0 mg/ml of tosylsul-

fonyl-phenylalanyl-chloromethyl-ketone [TPCK]-treated trypsin

[Sigma]) (1.6 ml per well in 6-well plates) at different tempera-

tures. Plaque assays were done in MDCK cells in the presence of

1.0 mg/ml TPCK-treated trypsin in agarose overlay medium (virus

growth medium containing 0.0015% DEAE-dextran hydrochlo-

ride [prepared from dextran of mean molecular weight 500,000;

Sigma] and 0.9% ultra-pure low-melting-point agarose [Invitro-

gen]), as reported previously [47]. After incubation at 37uC for

60 h the plaques were visualized by staining with 0.1% crystal

violet solution containing 10% formaldehyde.

Pathogenicity and transmission experimentsBaseline body weight and temperature were documented before

inoculation or contact exposure. Four donor ferrets per virus were

housed in the lower cages of isolators (configured as shown in

Fig. 1A) in ABSL2+ facilities. Air was uniformly circulated at 52 to

57 air changes per hour. Ambient temperature and relative

humidity were maintained. The donor ferrets were lightly

anesthetized with isoflurane and inoculated with 106 pfu of virus

in 0.5 ml PBS (250 ml per nostril). The next day (day 1 p.i.), two

donor ferrets were moved into separate cages, each containing one

naı̈ve direct contact (co-housed) ferret (Fig. 1A). For respiratory

droplet transmission assay, two naı̈ve ferrets were housed

separately in cages adjacent to the donor ferrets but separated

by double-layered (3 inches apart) grills to allow unobstructed

airflow but prevent direct contact. A borazine gun (Zero Toys,

Concord, MA) was used to ensure smooth air flow from the left

cages to the right cages within each isolator. The donor and

recipient ferrets remained housed together from day 1 p.i to day

20 p.i. Weight, temperature, and clinical signs (sneezing, lethargy,

and ruffled fur) were recorded every other day for 14 days. Nasal

washes were collected on days 2 (donors only), 4, 6, and 8 p.i. by

North American TRS Influenza A Viruses in Ferrets

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flushing nostrils with total 1.0 ml PBS, and pfu titers were

determined in MDCK cells. The two donor animals remaining in

the lower cages (Fig. 1A) were euthanized on day 5 p.i. for

histopathology of the lung and virus titration of the nasal

turbinates, trachea (upper and lower), and lung.

HistopathologyLung tissue was collected from control (un-inoculated) and

virus-inoculated ferrets on day 5 p.i., fixed in 10% neutral

buffered formalin, and embedded in paraffin. 5-mm sections were

stained with hematoxylin and eosin and examined by microscopy

in a blinded fashion. Histopathology was examined separately for

the bronchi, bronchioles, alveoli and alveolar interstitial walls of

each lung lobe.

Serologic testsSerum samples were collected from ferrets at day 20 p.i., treated

for 18 h at 37uC with receptor-destroying enzyme, heat-inactivat-

ed at 56uC for 30 min, and tested by HI assay with 0.5% packed

chicken red blood cells as described previously [48].

GenBank virus sequence accession numbersCY058484-91, CY098465-72, CY098473-80, CY098481-88,

CY098489-96, CY098497-504, CY098505, CY098506-12, and

CY098513-20 for A/Brisbane/59/2007 (H1N1), A/swine/MN/

1192/2001 [H1N2], A/swine/NC/47834/2000 [H1N1], A/

swine/MN/6998/2003 [H1N1], A/swine/MN/5763/2003

[H1N2], A/sw/Italy/1369-7/1994 [H1N1], A/Mexico/4482/

2009 (H1N1), A/swine/NC/38448-1/2005 [H1N1], and A/

swine/NC/18161/2002 [H1N1], respectively.

Supporting Information

Figure S1 Phylogenetic tree of the A) HA and B) NA gene

segments based on nucleotide sequences from GenBank. Evolu-

tionary history was inferred by using the neighbor-joining method.

The percentage of replicate trees in which the associated taxa

clustered in the bootstrap test (500 replicates) are shown next to

the branches. Only bootstrap values .60 are shown. The tree is

drawn to scale, with branch lengths indicating the evolutionary

distances used to infer the phylogenetic tree. The evolutionary

distances (the number of base substitutions per site) were

computed by using the Kimura 2-parameter method. All

ambiguous positions were removed for each sequence pair

(pairwise deletion option). Evolutionary analyses were conducted

by using the MEGA5 program. sw, swine; Eu sw, Eurasian avian-

like swine; hu, human; pan, pandemic. N, Viruses used in this

study.

(EPS)

Acknowledgments

We thank Mariette F. Ducatez for advice on phylogenetic analysis and

David S. Carey, Sharon Lokey, Henju Marjuki, Gururao Desai, Heather

Forrest, and Angela Ferguson for assistance with animals in the ABSL2+laboratory. We thank Dr. Christopher W. Olsen, Department of

Pathobiological Sciences, University of Wisconsin–Madison, for providing

A/sw/IN/9K035/99 (H1N2) virus. We also thank Sharon Naron for

editing the manuscript and Julie Groff for assisting in preparation of the

figures.

Author Contributions

Conceived and designed the experiments: SB RGW RJW. Performed the

experiments: SB PSK TPF JF JCT PS DW JER. Analyzed the data: SB

RGW RJW JER. Contributed reagents/materials/analysis tools: JER.

Wrote the paper: SB RGW RJW GAE MG. Conducted experiments with

ferrets: SB PSK TPF JF JCT PS DW. Sequenced the TRS viruses: SB PSK

TPF. Isolation and initial characterization of the TRS viruses: GAE MG.

Ferret lung pathology and data analysis: JER.

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